Basal laminae (basement membranes) are sheet-like, cell-associated extracellular matrices that play a central role in cell growth, tissue development, and tissue maintenance. They are present in virtually all tissues, and appear in the earliest stages of embryonic development.
Basal laminae are central to a variety of architectural and cell-interactive functions (See for example, Malinda and Kleinman, Int. J. Biochem. Cell Biol. 28:957-959 (1996); Aumailley and Krieg, J. Invest. Dermatology 106:209-214 (1996)). For example:                1. They serve as architectural supports for tissues, providing adhesive substrata for cells.        2. They create perm-selective barriers between tissue compartments that impede the migration of cells and passively regulate the exchange of macromolecules. These properties are illustrated by the kidney glomerular basement membrane, which functions as an important filtration structure, creating an effective blood-tissue barrier that is not permeable to most proteins and cells.        3. Basal laminae create highly interactive surfaces that can promote cell migration and cell elongation during embryogenesis and wound repair. Following an injury, they provide a surface upon which cells regenerate to restore normal tissue function.        4. Basal laminae present information encoded in their structure to contacting cells that is important for differentiation and tissue maintenance. This information is communicated to the cells through various receptors that include the integrins, dystroglycan, and cell surface proteoglycans. Signaling is dependent not only on the presence of matrix ligands and corresponding receptors that interact with sufficient affinities, but also on such topographical factors as ligand density in a three-dimensional matrix “landscape”, and on the ability of basal lamina components to cluster receptors. Because these matrix proteins can be long-lived, basal laminae create a “surface memory” in the basal lamina for resident and transient cells.        
The basal lamina is largely composed of laminin and type IV collagen heterotrimers that in turn become organized into complex polymeric structures. To date, six type IV collagen chains and at least twelve laminin subunits have been identified. These chains possess shared and unique functions and are expressed with specific temporal (developmental) and spatial (tissue-site specific) patterns.
Laminins are a family of heterotrimeric glycoproteins that reside primarily in the basal lamina. They function via binding interactions with neighboring cell receptors, and by forming laminin networks, and they are important signaling molecules that can strongly influence cellular function. Laminins are important in both maintaining cell/tissue phenotype as well as promoting cell growth and differentiation in tissue repair and development.
Laminins are large, multi-domain proteins, with a common structural organization. The laminin molecule integrates various matrix and cell interactive functions into one molecule.
A laminin molecule is comprised of an α-, β-, and γ-chain subunit joined together through a coiled-coil domain. Within this structure are identifiable domains that possess binding activity towards other laminin and basal lamina molecules, and membrane-bound receptors. Domains VI, IVb, and IVa form globular structures, and domains V, IIIb, and IlIa (which contain cysteine-rich EGF-like elements) form rod-like structures (Kamiguchi et al., Ann. Rev. Neurosci. 21:97-125 (1998)). Domains I and II of the three chains participate in the formation of a triple-stranded coiled-coil structure (the long arm).
Table 1 shows the individual chains that each laminin type is composed of:
TABLE 1Known laminin family members ProteinChains Laminin-1α1β1γ1Laminin-2α2β1γ1Laminin-3α1β2γ1Laminin-4α2β2γ1Laminin-5α3β3γ2Laminin-6α3β1γ1Laminin-7α3β2γ1Laminin-8α4β1γ1Laminin-9α4β2γ1Laminin-10α5β1γ1Laminin-11α5β2γ1Laminin-12α2β1γ3
Four structurally-defined family groups of laminins have been identified. The first group of five identified laminin molecules, including laminin 10 all share the β1 and γ1 chains, and vary by their α-chain composition (α1 to α5 chain). The second group of five identified laminin molecules all share the β2 and γ1 chain, and again vary by their α-chain composition. The third group of identified laminin molecules has one identified member, laminin 5, with a chain composition of α3β3γ2. The fourth group of identified laminin molecules has one identified member, laminin 12, with the newly identified γ3 chain (α2β1γ3).
Some progress has been made in elucidating the relationship between domain structure and function (See, for example, Wewer and Engvall, Neuromusc. Disord. 20 6:409-418 (1996)). The overall sequence similarity among the homologous domains in different chains varies, but it is highest in domain VI (thought to play a key role in laminin polymerization), followed by domains V (possibly involved in protein-protein interactions) and III (entactin/nidogen binding; possible cell adhesion sites), and is lowest in domains I, II (both thought to be involved in intermolecular assembly, and containing possible cell adhesion sites), and G. Not all domains are present in all 3 types of chains. The globular G domain (thought to be involved in cell receptor binding) is present only in the α chains. Other domains may not be present in all chains within a certain chain type. For example, domain VI is absent from α3, α4, and γ2 chains (Wewer and Engvall, 1996).
As a result of their large size (>600 kD) and unique structure, the laminin molecules can be resolved in the electron microscope (Wewer and Engvall, 1996). Typically, laminins appear as cross-shaped molecules in an EM. The three short arms of the cross represent the amino terminal portions of each of the three separate laminin chains (one short arm per chain). The long arm of the cross is composed of the C-terminal parts of the three chains, which together form a coiled coil structure (Wewer and Engvall, 1996). The long arm ends with the globular G domain.
The coiled-coil domain of the long arm is crucial for assembly of the three chains of laminin (Yurchenco et al., Proc. Natl. Acad. Sci. 94:10189-10194 (1997)). Disulfide bonds bridge and stabilize all three chains in the most proximal region of the long arm and join the β and γ chains in the most distal region of the long arm.
A model of laminin receptor-facilitated self-assembly, based on studies conducted with cultured skeletal myotubes and Schwann cells, predicts that laminins bind to their receptors, which freely diffuse in a fluidic membrane, when ligand-free. Receptor engagement forces the laminins into a high local two-dimensional concentration, facilitating their mass-action driven assembly into ordered surface polymers. In this process, the engaged receptors are also reorganized, accompanied by cytoskeletal rearrangements (Colognato, J. Cell Biol. 145:619-631 (1999)). This reorganization activates the receptors, causing signal transduction with the alteration of cell expression, shape and/or behavior. The evidence is that laminins must possess both cell-interacting and architecture-forming sites, which are located in different protein domains and on different subunits.
One class of laminin receptors are the integrins, which are cell surface receptors that mediate many cell-matrix and cell-cell interactions. Integrins are heterodimers, consisting of an α and a β subunit. 16 α- and 8 β-subunits are known, and at least 22 combinations of α and β subunits have been identified to date. Some integrins have only one or a few known ligands, whereas others appear to be very promiscuous. Binding to integrins is generally of low affinity, and is dependent on divalent cations. Integrins, activated through binding to their ligands, transduce signals via kinase activation cascades, such as focal adhesion and mitogen-activated kinases. Several different integrins bind different laminin isoforms more or less specifically (Aumailley et al., In The Laminins, Timpl and Ekblom, eds., Harwood Academic Publishers, Amsterdam. pp. 127-158 (1996)).
Laminin isoforms are expressed in tissue-specific and developmentally regulated patterns and they play significant roles in adhesion, migration, proliferation and differentiation of many cell types (Timpl, R., and Brown, J. C. (1994) Matrix Biol. 14(4), 275-81.; Ekblom, P., Timpl, R. (ed) (1996) The laminins Vol. 2. Cell Adhesion & Communication. Edited by Goridis, C., Harwood Academic Publishers GmbH, Amsterdam; Sorokin, L. M., et al. (1997) Dev. Biol. 189(2), 285-300.; Aumailley, M., and Smyth, N. (1998) J. Anat. 193(Pt 1), 1-21).
The laminin α5 chain, a component of laminin-10 (α5β1γ1) and laminin-11 (α5β2γ1), is expressed widely in adult tissues including placenta, heart, lung, skeletal muscle, kidney, and pancreas (Sorokin, L. M., et al. (1997) Dev. Biol. 189(2), 285-300; Patton, B. L., et al. (1997) J. Cell Biol. 139(6), 1507-21; Miner, J. H., et al. (1997) J. Cell Biol. 137(3), 685-701; Miner, J. H., et al. (1995) J. Biol. Chem. 270(48), 28523-6; Sorokin, L. M., et al. (1997) Dev. Dyn. 210(4), 446-62). Embryos lacking laminin α5 exhibit several developmental abnormalities, such as exencephaly and syndactyly, as well as dysmorphogenesis of the placental labyrinth and die late in embryogenesis (Miner, J. H., et al. (2000) Dev. Biol. 217(2), 278-89; Miner, J. H., et al. (1998) J. Cell Biol. 143(6), 1713-23). Laminin α5 chain-containing isoforms may therefore be important in placental endothelial cell migration and blood vessel branching, and in formation of proper basal laminae.
Integrin-mediated recognition of ECM molecules results in intracellular signaling that affects a range of cell behaviors (Clark, E. A. et al., Science 268(5208), 233-9 (1995)). In endothelial cells, these signals affect focal adhesions and cytoskeletal organization. Therefore, integrin-mediated endothelial cell recognition of laminin and other BM molecules may determine cell-to-matrix adhesiveness and mediate signals that are essential for the maintenance and normal functioning of blood vessels (Davis, G. E. et al., Exp. Cell Res. 216(1), 113-23 (1995); Dejana, E. et al., Kidney Int. 43(1), 61-5 (1993); and Shattil, S. J. et al., J. Clin. Invest. 100(11 Supp1), S91-5 (1997)). Laminin-8 and laminin-10 are secreted by endothelial cells, and are major components of the subendothelial basement membrane (Sorokin, L. M. et al., Dev. Biol. 189(2), 285-300 (1997); livanainen, A. et al., J. Biol. Chem. 272(44), 27862-8 (1997); Patton, B. L. et al., J. Cell Biol. 139(6), 1507-21 (1997), Miner, J. H. et al., J. Cell Biol. 137(3), 685-701 (1997); Sorokin, L. et al., Eur. J. Biochem. 223(2), 603-10 (1994); and Tokida, Y. et al., J. Biol. Chem. 265(30), 18123-9 (1990)).
There have been no reports of isolated laminin 10 that is free of other laminin chains. Studies on the function of laminin-10 have frequently used commercial preparations, which are normally prepared using proteolytic digestion and subsequent immunoaffinity chromatography resulting in a truncated mixture of α5-chain containing laminin isoforms (Sixt, M. et al., J. Biol. Chem. 276(22), 18878-87 (2001)). Attempts to purify laminin 10 from cell sources by affinity chromatography using laminin chain antibodies have been unsuccessful in eliminating, for example, laminin β2 chain, which is a component of laminin 11. (See, for example, Sixt, M. et al., J. Biol. Chem. 276(22), 18878-87 (2001)) Thus, such preparations represent a mixture of laminin 10 and laminin 11.
Despite the broad tissue distribution of the laminin α5 chain and laminin 10, the full length human laminin α5 chain sequence is not known, nor is there a means to isolate laminin 10 away from other laminins, nor has a means for recombinant expression of laminin 10 previously been developed. Isolated laminin 10 would have numerous research and therapeutic purposes including, but are not limited to, treating injuries to vascular tissue, promoting cell attachment and migration, ex vivo cell therapy, improving the biocompatibility of medical devices, and preparing improved cell culture devices and media.
Thus, there is a need in the art for isolated laminin-10 for research and therapeutic purposes, and methods for making isolated laminin 10.